1. Field of the Invention
The present invention relates to an anti-pathogen system that kills or injures pathogen-infected cells by introducing into the cells a fusion protein comprising a protein transduction domain and a cytotoxic domain. The cytotoxic domain is capable of being specifically activated in cells infected by the pathogen. Further provided are specified transduction domains that enhance transduction capacity of the fusion protein. The present invention has a variety of uses such as killing or injuring cells infected by one or more pathogenic viruses or plasmodia.
2. Background
A variety of pathogens infect mammals, particularly primates such as humans. For example, certain viruses, bacteria, fungi, yeasts, worms, plasmodia, and protozoa are recognized human pathogens. See e.g., Harrison""s Principles of Internal Medicine, 12th ed. McGraw-Hill, Inc. (1991).
Pathogens often kill or injure cells by mechanisms that manifest morphological characteristics. For example, pathogen-infected cells undergoing apoptosis or necrosis exhibit readily identifiable cellular changes.
There has been progress toward understanding cell proteins and particularly enzymes, involved in apoptosis. For example, certain cell proteases such as caspases (i.e. cysteinyl aspartate-specific proteases), C. elegans ced-3 and granzyme B have been implicated in apoptosis. Nucleic acid sequences encoding several capsases and proteolytic substrates for same are known. For example, caspase-3 (i.e. CPP32) has been particularly well-studied. See e.g., Thompson, C. B. Science, 267:1456 (1995); and Walker, N. P. C. et al. Cell, 78:343 (1994).
There have been related attempts to identify proteins involved in necrosis. For example, necrosis is thought to follow expression of certain DNA viruses such as herpes viruses.
Pathogens often induce synthesis of certain proteins, particularly enzymes such as proteases. It is likely that nearly all pathogens require one or more specific proteases to complete a productive infection. For example, it is believed that the following exemplary human pathogens require expression of at least one pathogen-specific protease: cytomegalovirus (CMV), herpes simplex virus, e.g., type-1 (HSV-1); hepatitis virus, e.g., type C (HCV); certain plasmodia, e.g., P. falciparum; human immunodeficiency virus type 1 (HIV-1, also referred to as HTLV-III, LAV or HTLV-III/LAV); human immunodeficiency virus type 2 (HIV-2), Kaposi""s sarcoma-associated herpes virus (KSHV or human herpes virus 8), yellow fever virus, certain flaviviruses and rhinovirus.
Sometimes the proteases are encoded by the pathogen itself. In this instance, the proteases are often referred to as pathogen-specific proteases. For example, CMV, HCV, HIV-1, HIV-2, KSHV, and P. falciparum are representative of pathogens that encode pathogen-specific proteases. These proteases serve a variety of functions and can be nearly indispensable for a productive infection.
There has been some efforts to analyze particular pathogen specific proteases such as serine-type proteinases encoded by HCV, aspartic proteases (i.e. plasmepsins I and II) encoded by P. falciparum, and a maturational protease encoded by HSV-1. See e.g., Dilanni, C. L. et al., J. Biol. Chem., 268:2048 (1993); and Francis, S. E. et al., EMBOJ., 13:306 (1994).
In contrast, inducible expression of certain host cell proteases is believed to modulate productive infection by other pathogens. These host cell proteases are sometimes referred to as inducible host cell proteases. For example, bacterial infection of eukaryotes such as certain plants can induce expression of normally quiescent host cell proteases. Induction of the host cell proteases may be an attempt to damage the pathogen, thereby protecting the host cell from infection.
Infection by HIV viruses has attracted substantial attention. There is now almost universal agreement that the human family of these retroviruses are the etiological agent of acquired immune deficiency syndrome (AIDS) and related disorders. Productive infection by nearly all HIV viruses requires expression of certain HIV-specific proteases. See, for example, Barre-Sinoussi et al., Science, 220:868-871 (1983); Gallo et al., Science, 224:500-503 (1984).
There has been progress toward developing therapeutic agents to target pathogen infections such as HIV infections. One general approach has focussed on interrupting distinct stages of the pathogen infection. In particular, therapeutic agents have been developed to combat certain HIV specific enzymes such as reverse transcriptase (RT) and pathogen-specific proteases.
Other agents such as certain cytokines have been used in attempts to treat CMV and HSV infections.
Other proposed methods for treating pathogen infections relate to what has been referred to as xe2x80x9cintracellular immunizationxe2x80x9d. Briefly, the methods involve genetically modifying host cells in an attempt to render them incapable of supporting a productive infection. For example, it has been suggested that certain eukaryotic cells can be made immune to pathogen infection by using the method. See e.g., Baltimore, Nature, 335:7395 (1988); Harrison et al., Human Gene Therapy, 3:461 (1992); and U.S. Pat. No. 5,554,528 to Harrison et al.
A more specific form of genetic modification has been reported to involve administering gene constucts that encode cytotoxins. In this instance, the contructs are desigened so that the genes can express cytoxin once inside the cells.
However, the prior methods for treating pathogen infections have several limitations.
For example, methods that use a cytotoxin to kill cells have not always been successful. One explanation may relate to pleiotropic effects reported for many intracellular cytotoxins. Those effects can often complicate analysis of cell killing. Additionally, many gene constructs that encode a cytotoxin can exhibit undesirably high basal activities inside host cells. These problems can produce what is known as xe2x80x9cleakyxe2x80x9d cytotoxin expression, leading to death of infected and non-infected cells.
Other methods for treating pathogen infection have also had problems. For example, methods relying on use of a drug have not been completely effective. More particularly, subjects infected by aggressive or persistent pathogens often equire prolonged therapeutic intervention, sometimes over a period of months or even years. Proliferation of drug resistant pathogens is becoming increasingly problematic. Thus, the long-term value of the methods is controversial.
In particular, current treatment of HIV utilizes small inhibitory molecules that target HIV protease. However, emergence of resistant HIV strains is increasingly problematic. See e.g., Coffin, J. M., et al. Retroviruses, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1997); Kaplan, A. H. et al. Selection of multiple human immunodeficiency virus type 1 variants that encode viral proteases with decreased sensitivity to an inhibitor of the viral protease. Proc. Natl. Acad. Sci. USA 91: 5597 (1994); Condra, J. H. et al. In vivo emergence of HIV-1 variants resistant to multiple protease inhibitors. Nature 374: 569 (1995); Gulnik, S. V. et al. Kinetic characterization and cross-resistance patterns of HIV-1 protease mutants selected under drug pressure. Biochemistry 34: 9282 (1995); and Tisdale, M. et al. Cross-resistance analysis of human immunodeficiency virus type 1 variants individually selected for resistance to five different protease inhibitors, Antimicrob. Agents Chemother. 39:1704 (1995).
There has been recognition that the retroviral TAT protein may find use in certain therapuetic settings. TAT has been reported to transactivate certain HIV genes and it is believed to be essential for productive infection by most human HIV retroviruses. The TAT protein has been used to bring certain types of fusion proteins into cells. This process is generally referred to as transduction. See U.S. Pat. No. 5,652,122 to Frankel et al.; and Chen, L. L. et al., Anal. Biochem., 227:168 (1995).
However, use of TAT to transduce fusion proteins into cells has been associated with significant shortcomings.
For example, it has been difficult to maintain suitable levels of the fusion proteins inside cells. Attempts to overcome this problem have included administering large amounts of the fusion proteins to help maintain adequate intracellular levels. The need to administer large amounts of the fusion proteins may prevent or hinder widespread use of some therapeutic fusion proteins. For example, use of large amounts of some TAT fusion proteins may negatively impact viability of some host cells.
Further, it has been difficult to maintain many of the prior transducing fusion proteins in a therapeutically relevant conformation. As an illustration, it is believed that many prior TAT fusion proteins may partially or completely unfold during transduction. That unfolding has potential to significantly reduce or eliminate transduction in many instances.
Additionally, the need to correctly fold the prior transducing fusion proteins has complicated efforts to purify and store the proteins.
Further drawbacks have been reported to be associated with proteins fused to TAT or certain TAT fragments. These drawbacks relate to how TAT is believed to act inside cells. More specifically, there has been acknowledgement that TAT or the TAT fragments may confer certain biological characteristics to the fusion proteins. Some of these characteristics and particularly nuclear localization and RNA binding may not always be desirable. In particular, there has been concern that many TAT fusion proteins may be difficult to position outside the nucleus or away from RNA. See e.g., Dang et al., J. Biol. Chem., 264:18109 (1989); Calnan, B. J. et al., Genes Dev., 5:201 (1991) for a discussion of TAT-associated properties.
It would be desirable to have an anti-pathogen system that exhibits high transduction efficiency and can specifically deliver a cytotoxin to pathogen infected cells. It would be further desirable to have an anti-pathogen system that can deliver the cytotoxin as an essentially inactive molecule that can be activated by pathogen infected cells.
The present invention relates to an anti-pathogen system that exhibits high transduction efficiency and specifically kills or injures cells infected by one or more pathogens. In general, the anti-pathogen system includes a fusion molecule that comprises a transduction domain and a cytotoxic domain genetically and hence covalently linked together as an in-frame fusion molecule. The invention further relates to transduction domains that enhance the transduction efficiency of the fusion molecules. The anti-pathogen system is essentially inactive in uninfected cells but it is specifically activated in cells infected by the pathogen. Further provided are methods of using the anti-pathogen system to treat infection by a pathogen and particularly human pathogens such as certain viruses and plasmodia.
Preferred use of the anti-pathogen system entails that the pathogen infection induce at least one pathogen specific protease. Preferably, that protease is capable of specifically cleaving a target amino acid sequence. The target amino acid sequence is one component of the fusion molecule and it is sometimes referred to herein as a protease recognition or cleavage site. Specific cleavage of the protease recognition site cleaves the fusion molecule, generally at or near the cytotoxin domain, to form a cytotoxin. The cytotoxin so formed is specifically capable of killing or injuring cells infected by the pathogen.
Significantly, the present anti-pathogen system links formation of the cytotoxin to presence of the pathogen-induced protease, thereby providing highly focussed cytotoxic action to infected cells. Formation of the cytotoxin is minimized or eliminated in uninfected cells and in infected cells that keep the pathogen inactive. The anti-pathogen system is therefore capable of effectively and specifically discriminating between productively infected and uninfected cells.
The present anti-pathogen system has a number of important advantages. For example, it can be readily manipulated to respond to changes in pathogen serotype. That is, the anti-pathogen system can be specifically tailored to kill or injure cells infected by one or more pathogen strains. In contrast, prior methods of blocking infection and especially drug-based methods are not usually designed to respond to changes in pathogen serotype. This deficiency often results in uncontrolled growth of drug-resistant pathogen strains. As will become more apparent from the discussion that follows, the anti-pathogen system has capacity to harness production of one or more pathogen-induced protease to kill or injure cells infected by the pathogen serotype. In marked contrast, most prior drug-based methods merely attempt to inhibit pathogenic processes, e.g., by blocking activity of a pathogen gene product. The present anti-pathogen system is more flexible and can be used to reduce or even eliminate emergence of pathogen strains by specifically exposing infected cells to cytotoxin.
As an illustrative example of the flexibility of the present invention, the anti-pathogen system is particularly useful against emergence of HIV serotypes. For example, many patients infected by HIV manifest several viral strains. Conventional drug-based therapies usually attempt to block activity of an HIV enzyme such as RT or an HIV protease. The clinical outcome of such treatment is often emergence of a spectrum of HIV serotypes. It has been recognized that the HIV serotypes can develop partial or even complete resistance to the therapies. Even so-called xe2x80x9ccocktailxe2x80x9d therapies employing multiple anti-HIV drugs have been problematic. In contrast, the anti-pathogen system of the present invention is highly flexible and can be adapted to kill or injure cells that produce the HIV serotypes by employing HIV proteases. Significantly, the anti-pathogen system is also formated to meet an increase in the activity of those HIV proteases or an increase in the number of infected cells with enhanced activation of the system.
The flexibility of the present anti-pathogen system arises in part because it can be tailored to kill or injure cells infected by nearly any number of HIV serotypes. Thus it is possible in accord with the present invention to format the anti-pathogen system to combat one or more HIV strains in a particular patient. This feature is highly useful in several respects. For example, it provides a specific method of fighting an HIV infection in a single patient without resorting to administration of potentially harmful or ineffective drugs. Significantly, the anti-pathogen system can be formatted to be effective at nanomoler doses or less. This low level of anti-viral activity is significantly lower than many present drug-based therapies. This feature of the invention positively impacts patient tolerance for the anti-pathogen system.
Further, the present anti-pathogen system is fully compatible with recognized anti-HIV therapies such as those using a xe2x80x9ccocktailxe2x80x9d format (ie. combination of anti-HIV drugs) to kill or injure infected cells.
In particular embodiments of the present invention, the anti-pathogen system is employed to reduce or eliminate emergence of HIV serotypes by exploiting the HIV protease produced by the virus.
Illustrative fusion proteins that kill HIV-infected cells are provided in Examples 11 and 12 below.
In addition, the present anti-pathogen system is capable of transducing unexpectedly large fusion molecules into cells. In particular, it has been discovered that the anti-pathgen system accomadates misfolded (i.e. partially or completely unfolded) fusion molecules and provides for efficient transduction of those molecules into cells. In particular, it is believed that the anti-pathogen system is compatible with misfolded fusion molecules having a molecular weight in the range of about 1 to about 500 kDa or more. The anti-pathogen system therefore is widely applicable to transducing a large spectrum of fusion molecules into cells.
More specifically, the ability to transduce misfolded fusion molecules has substantial advantages over prior transduction methods. For example, it has been found that misfolded fusion proteins used in accord with this invention significantly enhance transduction efficiency sometimes by as much as about 10 fold or greater. In addition, by misfolding the fusion proteins, it has been found that it is possible to optimize the amount of the fusion molecules inside cells. Preparation and storage of the fusion molecules are also positively impacted by the misfolding.
As discussed, the present anti-pathogen system is flexible. For example, it is not limited to any particular type of pathogen or cell provided that the pathogen is capable of inducing at least one specified protease in that cell. The protease can be a pathogen-induced or host cell induced protease that is specifically induced (i.e. synthesized or activated) in response to the infection. However, the specified protease must be capable of cleaving the protease recognition site on the fusion molecule to activate the cytotoxin.
The present anti-pathogen system and methods of using same can be used in vitro or in vivo. Further, the order or number of components of the fusion molecule are not important so long as each component on the molecule is operatively linked and can perform specified functions for which it is intended.
The cytotoxin produced by the anti-pathogen system is preferably selected to kill or injure infected cells in the presence of one or more of cell proteases and usually the pathogen- or host cell induced proteases. Preferably, the cytotoxin can kill at least about 20%, 25%, 50%, 75%, 80%, or 90% of the cells and preferably up to about 95%, 98% or 100% of the cells infected by the pathogen as assayed by standard cell viability tests. A preferred viability test is a standard Trypan Blue exclusion assay although other assays may be used as needed. It is also preferred that the cytotoxin activity be limited to cells in which it is produced.
As noted previously, the present anti-pathogen system includes an in-frame fusion molecule. The fusion can be accomplished by conventional recombinant nucleic acid methods. If desired, the fusion can also be achieved by chemically linking the transducing protein to the cytotoxic domain according to conventional methods described-below.
In general, the transduction domain of the fusion molecule can be nearly any synthetic or naturally-occurring amino acid sequence that can transduce or assist in the transduction of the fusion molecule. For example, transduction can be achieved in accord with the invention by use of a protein sequence and particularly an HIV TAT protein or fragment thereof that is covalently linked to the fusion molecule. Alternatively, the transducing protein can be the Antennapedia homeodomain or the HSV VP22 sequence, or suitable transducing fragments thereof such as those known in the field.
The type and size of the transducing amino acid sequence will be guided by several parameters including the extent of transduction desired. Preferred sequences will be capable of transducing at least about 20%, 25%, 50%, 75%, 80% or 90% of the cells of interest, more preferably at least about 95%, 98%% and up to about 100% of the cells. Transduction efficiency, typically expressed as the percentage of transduced cells, can be determined by several conventional methods such as those specific microscopical methods discussed below (e.g., flow cytometric analysis).
Additionally preferred transducing sequences will manifest cell entry and exit rates (sometimes referred to as k1 and k2, respectively) that favor at least picomolar amounts of the fusion molecule in the cell. The entry and exit rates of the amino acid sequence can be readily determined or at least approximated by standard kinetic analysis using detectably-labeled fusion molecules. Typically, the ratio of the entry rate to the exit rate will be in the range of from between about 5 to about 100 up to about 1000.
Particularly are transducing amino acid sequences that include at least a peptide featuring substantial alpha-helicity. It has been discovered that transduction is optimized when the transducing amino acid sequence exhibits significant alpha-helicity. Also preferred are those sequences having basic amino acid residues that are substantially aligned along at least one face of the peptide. Typically such preferred transduction sequences are synthetic protein or peptide sequences.
More preferred transducing amino acid sequences are referred to as class I transducing domains or like term and include a strong alpha helical structure with a trace of arginine (Arg) residues down the helical cylinder.
In one embodiment, the class I transducing domain is a peptide is represented by the following general formula: B1-X1-X2-X3-B2-X4-X5-B3; wherein B1, B2, and B3 are each independently a basic amino acid, the same or different; and X1, X2, X3, X4 and X5 are each independently an alpha-helix enhancing amino acid the same or different.
In another embodiment, the class I transducing peptide is represented by the following general formula: B1-X1-X2-B2-B3-X3-X4-B4; wherein B1, B2, B3, and B4 are each independently a basic amino acid, the same or different; and X1, X2, X3, and X4 are each independently an alpha-helix enhancing amino acid the same or different.
Additionally preferred transducing peptides are often referred to herein as xe2x80x9cclass IIxe2x80x9d domains or like terms. These domains generally require basic residues, e.g., lysine (Lys) or arginine (Arg), preferably arginine (Arg), and further including at least one proline (Pro) residue sufficient to introduce xe2x80x9ckinksxe2x80x9d into the domain.
In one embodiment, the class II domain is a peptide represented by the following sequence: X-X-Rxe2x80x94X-(P/X)-(B/X)-B-(P/X)-X-B-(B/X), wherein X is any alpha helical promoting residue, preferably alanine; P/X is either proline or X as previously defined; B is a basic amino acid residue, e.g., arginine (Arg) or lysine (Lys), preferably arginine (Arg); R is arginine (Arg) and B/X is either B or X as defined above.
Additional transducing sequences in accord with this invention include a TAT fragment that comprises at least amino acids 49 to 56 of TAT up to about the full-length TAT sequence. A preferred TAT fragment includes one or more amino acid changes sufficient to increase the alpha-helicity of that fragment. In most instances, the amino acid changes introduced will involve adding a recognized alpha-helix enhancing amino acid. Alternatively, the amino acid changes will involve removing one or more amino acids from the TAT fragment the impede alpha helix formation or stability. In more specific embodiment, the TAT fragment will include at least one amino acid substitution with an alpha-helix enhancing amino acid. Preferably the TAT fragment will be made by standard peptide synthesis techniques although recombinant DNA approaches may be preferred in some cases.
Additional transduction proteins of this invention include the TAT fragment in which the TAT 49-56 sequence has been modified so that at least two basic amino acids in the sequence are substantially aligned along at least one face of the TAT fragment and preferably the TAT 49-56 sequence. In one embodiment, that alignment is achieved by making at least one specified amino acid addition or substitution to the TAT 49-56 sequence. Illustrative TAT fragments include at least one specified amino acid substitution in at least amino acids 49-56 of TAT which substitution aligns the basic amino acid residues of the 49-56 sequence along at least one face of the segment and preferably the TAT 49-56 sequence.
Additional transduction proteins in accord with this invention include the TAT fragment in which the TAT 49-56 sequence includes at least one substitution with an alpha-helix enhancing amino acid. In one embodiment, the substitution is selected so that at least two basic amino acid residues in the TAT fragment are substantially aligned along at least one face of that TAT fragment. In a more specific embodiment, the subitution is chosen so that at least two basic amino acid residues in the TAT 49-56 sequence are substantially aligned along at least one face of that sequence.
Additionally provided are chimeric transducing proteins that include parts of at least two different transducing proteins. For example, chimeric transducing proteins can be formed by fusing two different TAT fragments, e.g., one from HIV-1 and the other from HIV-2. Alternatively, other transducing proteins can be formed by fusing a desired transducing protein to heterologous amino acid sequences such as 6XHis, (sometimes referred to as xe2x80x9cHISxe2x80x9d), EE, HA or Myc.
As noted above, the fusion molecule of the present invention also includes a fused cytotoxic domain. In general, the cytotoxic domain includes a potentially toxic molecule and one or more specified protease cleavage sites. By the term xe2x80x9cpotentially toxicxe2x80x9d is meant that the molecule is not significantly cytotoxic to infected or non-infected cells (preferably less than about 30%, 20%, 10%, 5%, 3%, or 2% cell mortality as assayed by standard cell viability tests. More preferred is 1% or less cell mortality) when present as part of the cytotoxic domain. As noted above, the protease cleavage sites are capable of being specifically cleaved by one or more than one of the proteases induced by the pathogen infection.
In particular, the protease cleavage sites are selected to remain essentially uncleaved in uninfected cells, thereby maintaining the cytotoxic domain in an inactive state. These protease cleavage sites may also be selected to remain essentially uncleaved in cells in which the pathogen is inactive. However, in the presence of a specified pathogen-induced or host cell induced protease, the protease cleavage sites are specifically cleaved to produce a cytotoxin from the potentially toxic molecule. That is, cleavage of the protease sites releases the cytotoxic domain from the fusion molecule, thereby forming an active cytotoxin. The one or more protease cleavage sites are generally positioned in the cytotoxic domain to optimize release of all or part of the domain from the fusion protein and to enhance formation of the cytotoxin.
More preferred protease cleavage sites are selected so as not to be cleaved by a protease normally associated with an uninfected cell. These proteases have been generically referred to as xe2x80x9chousekeepingxe2x80x9d proteases and are well known.
Protease cleavage sites are sometimes referred to herein as xe2x80x9cpathogen-specificxe2x80x9d cleavage sites to denote capacity to be specifically cleaved by one or more proteases induced by the pathogen infection. The protease cleavage sites are xe2x80x9cresponsivexe2x80x9d to a pathogen (or more than one pathogen) insofar as cleavage of those sites releases the cytotoxin domain from the fusion molecule, thereby activating the cytotoxin.
The cytotoxic domain can include one or more of a variety of potentially toxic molecules provided that it can be released from the fusion molecule as discussed. An illustrative cytotoxic domain for use in the fusion molecules includes an immature enzyme. These immature enzyme is sometimes referred to as zymogen, proenzyme, preproenzyme or simply as xe2x80x9cpre-xe2x80x9d xe2x80x9cpre-proxe2x80x9d or xe2x80x9cpro-xe2x80x9d forms of more mature enzyme. Preferred zymogens can be specifically activated to a cytotoxin (ie. a cytotoxic enzyme) by site-specific proteolysis at one or more naturally-occuring protease cleavage sites on the zymogen. The zymogens can be further processed in some instances by self-proteolysis.
Particularly, a cytotoxic domain that includes a preferred zymogen will include one or more specified protease cleavage sites that have been added within and/or around the zymogen. The cleavage sites are optionally positioned to facilitate release and processing of the zymogen to a mature or more mature cytotoxic enzyme.
In particular, the addition of the protease cleavage sites to the zymogens can be supplative with respect to the naturally-occurring protease cleavage sites in that zymogen. However it is preferred that the cleavage sites be substituted for one or more of the naturally-occurring cleavage sites. In this embodiment, the substituted protease cleavage sites in the zymogen are capable of being specifically cleaved by one or more pathogen-specific proteases. It has been found that by partially or completely substituting the naturally-occurring protease cleavage sites of the zymogen with one or more pathogen responsive cleavage sites, maturation of the zymogen into a cytotoxin is brought under substantial or complete control by the pathogen infection.
A variety of specific zymogens are suitable for inclusion in the cytotoxic domain as discussed below. Active forms of those zymogens generally include bacterial toxins and particularly exotoxins, plant toxins, and invertebrate toxins including conotoxins, snake and spider toxins.
Further contemplated cytotoxic domains include known proteins with potential to exert genetically dominant characteristics. That is, the proteins can be specifically cleaved from the fusion protein and can subsequently override one or more cell functions such as cell replication. In this embodiment, the potentially dominant protein must not manifest the dominant characteristic (sometimes known as a dominant phenotype) until that protein is released from the fusion protein. Examples of potentially dominant proteins in accord with the invention include proteins that inhibit cell replication such as the retinoblastoma protein (Rb), p16 and p53.
Further contemplated cytotoxic domains include essentially inactive enzymes that have capacity to convert certain nucleosides or analogs thereof into a cytotoxin. In this embodiment, the cytotoxic domain will include one or more specified protease cleavage sites, that is preferably positioned to release the inactive enzyme from the fusion protein. Following the release, the enzyme converts the nucleoside or analog thereof into a cytotoxin. Examples of such enzymes include viral thymidine kinase and nucleoside deaminases such as cytosine deaminase. Also contemplated are cytotoxic domains comprising catalytically active fragments of the enzymes such as those generally known in the field.
The present anti-pathogen system provides a number of additional important advantages. For example, the anti-pathogen system unexpectedly accommodates misfolded fusion proteins. As will become more apparent from the discussion and examples which follow, that feature has been found to substantially boost levels of the fusion protein inside cells. Typically, a corresponding increase in the amount of administered fusion protein is not required. Without wishing to be bound to theory, it is believed that transduction of misfolded fusion molecules requires modest numbers of molecules and only a few of those need be refolded to manifest an effective cytotoxic effect. For example, it is believed that with certain preferred fusion proteins such as those described below in Examples 5-6, only about 10 to 100 correctly refolded fusion proteins are needed to kill or injure infected cells. Thus, the present invention can decrease or even eliminate the need to concentrate large number of cytotoxic molecules inside cells to achieve significant anti-pathogen activity.
In addition, it has been found that activity of the present anti-pathogen system is enhanced in many cases by mass action. More particularly, it has been found that specific cleavage of the cytotoxic domain can draw additional fusion molecules into infected cells. This feature can be particularly advantageous for those fusion proteins that include cytotoxic domains which are preferably administered in sub-optimal doses. In such instances, the fusion protein is specifically concentrated in infected cells, thereby increasing levels of the cytotoxin to lethal or near lethal levels. Importantly, the cytotoxin remains at sub-optimal levels in uninfected cells.
Still further advantages are provided with respect to particular fusion proteins of the invention that include the TAT fragment described above. For example, the cytotoxic domain of a protein fused to the TAT fragment need not be directed to the cell nucleus or to RNA. More specifically, the present fusion molecules are formatted to separate the cytotoxic domain from the TAT fragment inside infected cells, thereby avoiding unnecessary concentration of the protein in the nucleus or with RNA. It is recognized that in uninfected cells, such fusion proteins may be directed to the nucleus or to RNA. Thus, differential localization of the fusion protein in infected and non-infected cells can provide means of distinguishing such cells from one another, e.g., by inspection.
The anti-pathogen system of the invention can also positively impact certain drug-based anti-pathogen therapies. More specifically, cells infected by retroviruses and particularly HIV can harbor infectious particles for long periods of time, sometimes months or even years. Over this time, retroviruses can develop substantial resistance to most drugs, sometimes by changing one or only a few genomic sequences. It has been recognized that once the retroviruses become resistant to one class of drugs, such viruses can be become resistant to a spectrum of drugs. Thus, therapies using drug-based approaches are generally inflexible and do not readily adapt to presence of resistant viruses. Related concerns have been raised with respect to development of other resistant pathogen strains such as certain plasmodia.
In contrast, the present anti-pathogen systems kills or injures cells infected by pathogens regardless of pathogen capacity to acquire drug resistance. It is believed that development of drug resistant pathogens and particularly drug resistant HIV strains, is nearly impossible with the present anti-pathogen system due to the large number of protease cleavage sites that the system can accommodate. As an illustrative example, HIV virus has been reported to have about 8 to 10 such cleavage sites. In order to develop substantial resistance against the anti-pathogen system, which system could include one or more of these sites, that virus would have to modify those cleavage sites as well as the corresponding viral protease.
Accordingly, use of the present anti-pathogen system is expected to significantly reduce or even eliminate the presence of many pathogen resistant strains and particularly certain drug resistant HIV strains.
Additionally, the anti-pathogen system of the invention is compatible with a variety of drug-based therapies. Thus, the anti-pathogen system can be used as a sole active agent or in combination with one or more therapeutic drugs, e.g. to minimize or eliminate pathogens and particularly drug resistant pathogen strains.
Further provided are substantially pure fusion molecules of the invention.
The invention also provides nucleic acid sequences encoding the fusion proteins, particularly extrachromosomal DNA sequences organized as an autonomously replicating DNA vector.
The invention also provides methods for suppressing or eliminating infection by one or more pathogens in a mammal, particularly a primate such as a human. The methods more specifically include administering a therapeutically effective amount of the present anti-pathogen system. The methods further include treatment of a mammal that suffers from or is susceptible to infection by one or pathogens.
Preferred methods according to the invention for suppressing or eliminating infection by the one or more pathogens include providing the anti-pathogen system as an aerosol and administering same, e.g., through nasal or oral routes. Particularly contemplated are modes of administration which are specifically designed to administer the anti-pathogen system to lung tissue so as to facilitate contact with lung epithelia and enhance transfer into the bloodstream.
Also provided are methods of inducing apoptosis in a pre-determined population of cells in which the method comprises administering to the mammal such as a primate and particularly a human a therapeutically sufficient amount of the anti-pathogen system in the presence of one more pathogens.
The cell infected by one or more pathogens may be a cell maintained in culture, e.g., an immortalized cell line or primary culture of cells or tissue; or the cell can be part of a tissue or organ in vivo (e.g., lung). Thus, the present anti-pathogen system can be used in vitro and in vivo as needed.
The invention also provides substantially pure fusion molecules and particularly fusion proteins that in addition to the aforementioned transduction and cytotoxic domains may also include other components as needed. These components can be covalently or non-covalently linked thereto and may particularly include one or more polypeptide sequences. An added polypeptide sequence will sometimes be referred to herein as protein identification or purification xe2x80x9ctagxe2x80x9d. Exemplary of such tags are EE, 6Xhis, HA and MYC.
As discussed, it is preferred that the fusion proteins described herein by provided in misfolded form although in some instances it may be desirable to use properly folded fusion proteins. The misfolded fusion proteins are typically purified by chromatographic approaches that can be tailored if needed to purify a desired fusion molecule from cell components which naturally accompany it. Typically, the approaches involve isolation of inclusion bodies from suitable host cells, denaturation of misfolded fusion proteins, and use of conventional chromatographic methods to purify the fusion molecules. Expression of the misfolded fusion proteins in the inclusion bodies has several advantages including protecting the misfolded fusion protein from degradation by host cell proteases. In addition, by providing the fusion proteins in misfolded form, time-consuming and costly protein refolding techniques are avoided.
Further provided by the present invention are methods of making substantial quantities of the fusion molecules. Generally stated, the methods include expressing desired fusion molecules in suitable host cells, culturing the cells, and purifying the fusion molecules therefrom to obtain substantially pure fusion molecules. The methods can be used to express and purify a desired fusion protein on a large-scale (i.e. in at least milligram quantities) from a variety of implementations including roller bottles, spinner flasks, tissue culture plates, bioreactor, or a fermentor.
The present methods for isolating and purifying the fusion proteins of the invention are highly useful. For example, for a fusion protein exhibiting a desired killing or injuring activity, it is very useful to have methods for expressing and purifying the fusion proteins. It is particularly useful to have methods that can produce at the fusion proteins in large quantities, so that the fusion molecule can be made as one component of a kit suitable for medical, research, home or commercial use. Further, it is useful to have large-scale quantities of the fusion proteins available to simplify structural analysis, as well as for further purification and/or testing if desired.
The invention also features in vitro and in vivo screens to detect compounds with therapeutic capacity to modulate and preferably inhibit, proteins and especially proteases induced by a pathogen infection. For example, one method generally comprises infecting a desired cell with a pathogen, contacting the cell with a fusion protein of the invention, transducing the fusion protein, adding the compound to the cells and detecting cells killed or injured by the fusion protein. Efficacy of a particular compound can be readily evaluated by determining the extent of cell killing or injury as a function of concentration of the added compound.
Further provided are methods of suppressing a pathogen infection in a mammal, particularly a primate such as a human, comprising administering to the mammal a therapeutically effective amount of the anti-pathogen system. In one embodiment, the fusion protein includes a covalently linked protein transduction domain and a cytotoxic domain. The method includes transducing the fusion protein into cells of the mammal, cleaving the fusion protein sufficient to release the cytotoxic domain from the fusion protein, concentrating the cytotoxic domain in the cells; and producing a cytotoxin sufficient to suppress the pathogen infection in the mammal. Exemplary pathogens include but are not limited to retroviruses, herpesviruses, viruses capable of causing influenza or hepatitis; and plasmodia capable of causing malaria. Preferred cytotoxic domains and cytotoxins are described in more detail below.
In another embodiment of the method for suppressing the pathogen infection in the mammal, a prodrug is administered (e.g., a suitable nucleoside or analog thereof) and a cytotoxin is produced by contacting the prodrug with the concentrated cytotoxic domain.
Further provided by the present invention are fusion proteins that include covalently linked in sequence: 1) A TAT segment and particularly a protein transducing fragment thereof, and 2) a pathogen induced or host cell induced protease, e.g., HIV protease; or a catalytically active fragment thereof.
Additionally provided by the invention is an anti-pathogen system, wherein the fusion protein comprises covalently linked in sequence: 1) a transduction domain, 2) a first zymogen subunit, 3) a protease cleavage site, and 4) a second zymogen subunit. Also provided is an anti-pathogen system, wherein the transduction domain is TAT, the first zymogen subunit is p5 Bid, the protease cleavage site is an HIV protease cleavage site and the second zymogen subunit is p15 Bid.
The invention also provides an anti-pathogen system, wherein the fusion protein comprises covalently linked in sequence: 1) a transduction domain, 2) a first protease cleavage site, 3) first zymogen subunit, 3) a second protease cleavage site, and 4) a second zymogen subunit. Also provided is an anti-pathogen system, wherein the transduction domain is TAT, the first protease cleavage site is an HIV p7-p1 protease cleavage site, the first zymogen subunit is p17 caspase-3, the second protease cleavage site is an HIV p17-p24 protease cleavage site, and the second zymogen subunit is p12 caspase-3.
The present invention also provides a method of killing an HIV-infected cell. In one embodiment, the method includes contacting the cell with an effective dose of a fusion protein, wherein the fusion protein comprises covalently linked in sequence: 1) a transduction domain, 2) a first zymogen subunit, 3) a protease cleavage site, and 4) a second zymogen subunit; or 1) a transduction domain, 2) a first protease cleavage site, 3) first zymogen subunit, 3) a second protease cleavage site, and 4) a second zymogen subunit. The fusion protein can be administered in vitro or in vivo as needed. For example, the fusion protein can be administered in vivo to a mammal in need of such treatment, e.g., a primate and particularly a human patient infected by the HIV virus.
Other aspects of the invention are disclosed infra.